H+-Gated Ion Channel

ABSTRACT

The invention relates to an isolated or recombinant Na + /H +  exchanger comprising an isolated or recombinant Na + /H +  exchanger, particularly to the PBO-4 Na + /H +  exchanger. Also disclosed is an isolated or recombinant protein component of an H + -gated channel which can be affected by extracellular Ca 2+  concentration. In particular, the invention relates to PBO-5 and/or PBO-8 and/or a H | -gated channel composed of PBO-5 and PBO-8. The invention relates to compounds isolated from a vertebrate organism, wherein said compounds comprise at least a part of a H + -gated channel or Na + /H +  exchanger. The invention also relates to a method for identifying a component of a H + -gated channel in a vertebrate organism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/587,018(now abandoned), which is a National Phase Application of InternationalApplication No. PCT/US2005/013415, filed Apr. 20, 2005, which claimspriority to U.S. Provisional Application No. 60/563,939, filed Apr. 20,2004, the entirety of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant MH60997-02awarded by the National Institute of Health. The government has certainrights in the invention.

TECHNICAL FIELD

This invention relates to biotechnology, more particularly to one ormore members of the “cys-loop” ligand-gated ion channel superfamily,which are activated by a proton and/or H⁺/Na⁺ exchange proteins, andmethods of using the same.

BACKGROUND

Motor behaviors such as locomotion rely on precise signaling from thenervous system to coordinate various muscle activities. Theneuromuscular junction has been extensively studied and a considerableamount of information exists on how information is communicated acrossthe synapse between a motor neuron and a muscle cell. At the cellularlevel, depolarization of the motor neuron produces an action potentialthat is propagated to the presynaptic terminal. Depolarization causesthe opening of voltage-gated calcium channels at the presynapticterminal, resulting in an increase in intracellular calcium. In responseto local increases in calcium, neurotransmitter filled vesicles fusewith the presynaptic plasma membrane, releasing their contents into thesynaptic cleft. Postsynaptic ligand-gated ion channels on the musclebind the neurotransmitter, resulting in the opening of an integral ionchannel. Depending on an ion channel's permeability, the muscle iseither hyperpolarized or depolarized.

Chemical synaptic transmission mediates fast and slow communicationbetween cells, but it is not the only means of communication within thenervous system. Signals may also be conveyed nonsynaptically. In thiscase, a neurotransmitter or hormone is released at one site and slowlydiffuses to distant target sites that are not in contact with theoriginal site of release. This type of signaling is more suited toglobal, modulatory activities rather than functions requiring a rapidresponse. The interplay of synaptic and non-synaptic communicationultimately determines how a nervous system mediates particularbehaviors.

In C. elegans, synaptic transmission has been extensively studied at thegenetic, cellular and molecular levels. Most behaviors in the wormfollow a simple paradigm in which information is directed from thesensory neurons to motor neurons to muscle, via synaptic contacts.However, the defecation cycle in C. elegans is a unique behavior in thatit appears to be mediated by both synaptic, as well as nonsynaptictransmission (McIntire et al., 1993b; Thomas, 1990). Defecation in C.elegans is a stereotyped behavior that occurs every 50 seconds for thelife of the animal, and is characterized by the coordinated activationof three independent muscle contractions (Croll, 1975; Thomas, 1990).The cycle is initiated with a posterior body contraction, followed by ananterior body contraction and finalized by an enteric musclecontraction, which expels intestinal contents. A simplified geneticpathway to explain the defecation behavior was proposed by Jim Thomas(Thomas, 1990) (see, FIG. 1B). In this model, a clock mechanism keepstime independently of the motor program. At the appropriate interval,the clock first signals the posterior body contraction and then signalsthe common anterior body and enteric muscle contraction mechanism, whichultimately leads to activation of the individual muscle contractions(Liu and Thomas, 1994; Thomas, 1990) (FIG. 1B).

To determine the cellular basis of the defecation motor program,extensive cellular laser ablations have been performed. Based on thesestudies the motor neurons AVL and DVB were demonstrated to mediate theanterior body and enteric muscle contraction, but not posterior bodycontraction (McIntire et al., 1993b). The anterior body contraction ismediated by the motor neuron AVL, but the neurotransmitter that mediatesthis contraction is unknown (see, FIG. 1A). While AVL alone is requiredfor anterior body contraction, both the AVL and DVB motor neurons servea redundant function in activating the enteric muscles (see, FIG. 1A).Activation of the enteric muscles is GABA-dependent and is mediated bythe EXP-I receptor (Beg and Jorgensen, 2003; McIntire et al., 1993a;McIntire et al, 1993b).

Interestingly, no known neurons are required to maintain the clock orinitiate the posterior body contraction, suggesting that cycle timingand posterior body contraction are mediated by a non-neuronal mechanism(Liu and Thomas, 1994; McIntire et al., 1993b; Thomas, 1990) (see, FIG.1A). Furthermore, mutations that disrupt classical neurotransmission andsecretion do not affect the posterior body contraction. Taken together,these data suggest that posterior body contraction occurs through anon-neuronal mechanism that does not rely on classical or peptidergicneurotransmission.

Many genes have been identified that affect only specific aspects of thedefecation motor program. It has been demonstrated that timekeeping ofthe cycle is controlled by an endogenous clock that resides in theintestine (Dal Santo et al., 1999). The cycle time is set by theactivity of the itr-1 gene, which encodes an inositol triphosphate (IP3)receptor which mediates release of calcium from the smooth endoplasmicreticulum into the intestine every 50 seconds (Dal Santo et al., 1999).Mutations in the itr-1 gene slow down or eliminate the cycle, whileoverexpression accelerates the cycle. In the intestine, calcium levelsoscillate with the same period as the defecation cycle (50 seconds) andpeak calcium levels immediately precede the posterior body contraction(Dal Santo et al., 1999). Therefore, the frequency of intracellularcalcium release in the intestine, determines the frequency of thedefecation cycle.

It has been demonstrated that there is a one-to-one relationship betweenthe calcium spike in the intestine and the execution of the posteriorbody contraction (Dal Santo et al., 1999). Normally, motor behaviorssuch as muscle contraction are mediated by the nervous system. Sinceneuronal input is not required to initiate the posterior bodycontraction, these muscles could be directly activated by a Ca²⁻regulated signal from the intestine.

BRIEF SUMMARY

In accordance with the purpose of this invention, as embodied andbroadly described herein, this invention relates to an isolated orrecombinant Na⁺/H⁺ exchanger comprising an isolated or recombinantNa⁺/H⁺ exchanger. The invention further relates to the PBO-4 Na⁺/H⁺exchanger and Na⁺/H⁺ exchangers having at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,and/or 99% identitt to PBO-4 (SEQ ID NO:8). Optionally, the isolated orrecombinant Na^(|)/H^(|) exchanger functions in the intestine.

The invention also relates to isolated or recombinant protein componentof an H⁺-gated channel. The invention further relates to a H⁺-gatedchannel that is effected by extracellular Ca²⁺ concentration. Inparticular, the invention relates to PBO-5 (SEQ ID NOs: 1 and 2) and/orPBO-8 (SEQ ID NOs: 3-5) and/or a H⁺-gated channel composed of PBO-5 andPBO-8. In addition the invention relates to isolated and/or recombinantproteins having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% identity toPBO-5 and/or PBO-8. Further, the invention relates to a H^(|)-gatedchannel that is pH sensitive. The invention further relates to aH⁺-gated cation channel.

The invention relates to compounds isolated from a vertebrate organism,wherein said compounds comprise at least a part of a H⁺-gated channel orNa⁻/H⁺ exchanger. The invention further relates to a Na⁺/H⁺ exchangeridentified in a vertebrate organism.

The invention also relates to a method for identifying a component of aH⁺-gated channel in a vertebrate organism. For example, by screening avertebrate organism for the presence of a component of a H⁺-gatedchannel; identifying the component of the H⁺-gated channel; andconfirming that the component of the H⁺-gated channel is a component ofsaid H⁺-gated channel. Optionally, the method further comprisingisolating the component of said H⁺-gated channel.

The invention also relates to screening a component of a H⁺-gatedchannel for activation or inhibition by a candidate drug. The inventionalso relates to screening the component of a H⁺-gated channel forbinding to HEPES and/or identifying mutations which prevent binding toHEPES.

The invention further relates to a protein produced by a process ofscreening a vertebrate organism for the presence of a component of aH⁻-gated channel; identifying said component of said H⁺-gated channel;and confirming that said component of said H-gated channel is acomponent of said H⁺-gated channel.

The invention further relates to compounds according to the inventionidentified in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 illustrates the defecation cycle behavior and genetics of thedefecation cycle in C. elegans. FIG. 1A is a schematic drawing of thedefecation cycle. Every 50 seconds a cycle is initiated by the posteriorbody contraction (pBoc), followed by an anterior body contraction(aBoc), and completed by an enteric muscle contraction, (Emc), whichmediates expulsion of intestinal contents. Listed to the right are themotor neurons and neurotransmitters involved in each muscle contraction.FIG. 1B illustrates the genes involved and their affects on the cycle. A50 second clock independently keeps time of the cycle. Each step of thedefecation cycle can be specifically affected by mutation, demonstratingthat cycle timing and muscle activation do not rely on one another.Muscle activation of each step occurs independently of the others.However, the anterior body contraction and the enteric musclecontraction share a common mechanism upstream of muscle activation.

FIG. 2 illustrates the structure and molecular cloning of pbo-5. Thegenomic location of pbo-5 is the last predicted gene on chromosome V,approximately 1.2 kb from the telomere. The middle of FIG. 2 illustratesthe exon-intron structure of the pbo-5 gene. Positions of mutations inpbo-5 alleles are shown below the predicted protein structure. Trianglesrepresent the peptide signal, the dotted line is the disulfide-bond, thetransmembrane domains are labeled 1-4.

FIG. 3 shows the sequence alignment of PBO-5 and cholinergic subunits.The amino acid sequence for C. elegans PBO-5, PBO-8 (F11C7.1; SEQ IDNO:5), UNC-29, UNC-38, and human alpha 7 nicotinic acetylcholinereceptors subunits. Sequences were aligned using clustal X andidentities are shaded and boxed. The black and hollow filled trianglesdenote the signal peptide sequence for PBO-5 and PBO-8, respectively.The dotted line shows the invariant disulfide bond present in allligand-gated ion channels. Black bars denote the four transmembranedomains.

FIG. 4 shows the pbo-8 genomic structure and a phylogenetic tree. FIG.4(A) shows the exon-intron structure of pbo-8. The pbo-8 locus consistsof 13 exons that span 4.1 kb of the genome. FIG. 4(B) illustrate thatPBO-5 and PBO-8 represent novel ligand-gated ion channel subunits. ThePBO-5 and PBO-8 subunits cannot be categorized into one of the fourligand-gated ion channel families based on sequence analysis. Alignmentswere performed using clustal X and the bootstrap method with‘neighbor-joining’ search was used to create the tree. Bootstrap valuesof 1,000 replicates are indicated on the tree.

FIG. 5 shows the expression pattern of pbo-5. FIGS. 5A and 5B areconfocal images of animals expressing an integrated pbo-5::gfp fusiongene. The animals are oriented with anterior to the left, and posteriorto the right. GFP expression is observed in the most posterior musclecells (FIG. 5A) and in the neurons RIFL, RIFR, and RIS (FIG. 5B). FIG.5C is a schematic drawing to illustrate pbo-5 expression within thecontext of the entire animal.

FIG. 6 illustrates the structure and molecular cloning of pbo-8. The topof FIG. 6 A represents the exon-intron structure of pbo-8; below, thepredicted protein structure. The triangle represents the predictedsignal peptide cleavage site, the dotted line represents thedisulfide-bond, and the numbers 1-4 represent the four transmembranedomains. FIG. 6B shows the expression pattern of PBO-8. The left panelis a confocal image of an animal expressing a transcriptional pbo-5::gfpfusion gene. The right panel is a fluorescent image of an animalexpressing a transcriptional pbo-8::gfp fusion gene fusion. PBO-8expression is observed in the most posterior muscle cells, and hasoverlapping expression with PBO-5, suggesting PBO-8 may oligomerize withPBO-5 to form a functional receptor.

FIG. 7 shows a pH dose-response curve for PBO-5/PBO-8, which form aheteromultimeric H⁺-gated ion channel. PBO-5/PBO-8 expressing oocyteswere voltage-clamped at −60 mV and a series of test pH applications(7.2-5.0) were bath applied for 5 seconds. Each point represents themean current value normalized to the maximum and minimum values. ApH₅₀=6.83±0.01 and Hill coefficient of 9+0.66 were determined forPBO-5/PBO-8 receptors (n =18). Error bars represent s.e.m. Inset,representative traces of PBO-5/PBO-8 dose-response experiments.

FIG. 8 shows PBO-5/PBO-8 ion selectivity. FIG. 8A shows thecurrent-voltage (I-V) relationship of PBO-5/PBO-8 ion channels. Thereversal potential determined under control conditions was 10.18±0.80 mV(n=13). Inset, representative traces of an experiment. Note, the stronginward rectification of PBO-5/PBO-8 receptors. FIG. 8B shows Na⁺permeability. Na ions were replaced with the cation NMDG, in Na⁺ freeRinger's. There is a negative shift in reversal potential(E_(rev)=−82.90±8.13 mV (n=6)) and the inward current in nearlyabolished which suggests that Na is the primary charge carrier throughthe PBO-5/PBO-8 channel. FIG. 8C shows Cl⁻ permeability. Chloride ionswere replaced with the anion gluconate, in Cl⁻ free Ringer's. Thenegligible shift in reversal potential demonstrates that chloride ionsdo not underlie the ionic conductance through PBO-5/PBO-8 channels(ΔE_(rev)=0.25 mV, P>0.05, two-way ANOVA, (n=8).

FIG. 8D shows K⁺ permeability. Replacement of K⁺ for Na⁺ in theextracellular solution demonstrates PBO-5/PBO-8 ion channelsdiscriminate poorly between monovalent cations. Note the inward currentis still present and there was not significant shift in reversalpotential compared to control (P>0.05; two-way ANOVA, (n=4),demonstrating PBO-5/PBO-8 is a nonselective cation channel.

FIG. 9 shows calcium permeability. FIG. 9A shows the current-voltage(I-V) analysis of PBO-5/PBO-8 receptors under different Ca²⁻concentrations. Reversal potentials were measured under 1 mM Ca²⁺control and 3 mM and 10 mM test Ca²⁺ conditions. Increasingextracellular Ca²⁺ caused a positive shift in reversal potentialssuggesting Ca²⁺ is permeable to PBO-5/PBO-8 channels. Note the decreasedinward current as extracellular Ca increased. FIG. 9B shows Ca^(2|)permeability in Na^(|) free Ringers. To better resolve Ca^(2|)permeability, the I-V relationships where Ca²⁺ was the only relevantextracellular ion were determined. These data demonstrate PBO-5/PBO-8ion channels are Ca²⁺ permeable.

FIG. 10 shows that Ca²⁺ and H⁺ compete at the PBO-5/PBO-8 activationsite. FIG. 10A shows the results of normalized current amplitude versustest pH at four different extracellular Ca²⁺ concentrations. IncreasingCa²⁺ decreases the pH necessary for half-maximal activation. Currentswere normalized to the maximal value at pH 6.0 for each [Ca^(2|)]_(o)condition. FIG. 10B shows that a normalized current amplitude of test pHevoked responses at three Ca²⁺ concentrations. Ca²⁺ shifts activationbut increasing extracellular does not affect maximal activation. Currentwere normalized to the value at 1 mM Ca²⁺ for each pH tested.

FIG. 11 illustrates an exemplary embodiment for posterior bodycontraction in C. elegans. Every 50 seconds, a calcium spike occurs inthe intestine the correlates with the initiation of the posterior bodycontraction. First, IP₃ receptors localized to the smooth endoplasmicreticulum in the intestine are activated every 50 seconds. Activationresults in the intracellular rise of Ca²⁺ in the intestine. Second, Ca²⁺binds to the C-terminal calmodulin-binding domain of PBO-4, therebyactivating H⁺ transport activity. Third, PBO-4 transports H⁺ ions out ofthe intestine acidifying the pseudocoelomic space. Fourth, H⁺ ions bindand activate the PBO-5/PBO-8 receptors expressed in posterior body wallmuscle, thereby allowing Na⁺ influx into the cell causing adepolarization and subsequent muscle contraction.

FIG. 12 illustrates the genetic mapping and molecular cloning of pbo-4.FIG. 12(A) illustrates the pbo-4 locus, which maps between daf-12 andegl-15 on the X chromosome. This region contains the cosmids T21B6 andK09C8. Rescue of the posterior body contraction defect by cosmid orsubclone is indicated. Below this is an exon-intron structure of pbo-4.Mutations are indicated, black bars denote the extent of deletionalleles within the locus. FIG. 12(B) illustrates the predicted PBO-4protein domains. The predicted protein consists of a signal peptide, 12transmembrane domain, a re-entrant loop between transmembrane domains 9and 10 and an intracellular carboxy-terminal tail that contains apredicted calmodulin binding site.

FIG. 13 shows a sequence alignment of PBO-4 to human Na⁻/H⁺ exchangers.Deduced polypeptide sequence of PBO-4 aligned with human NHE1(P19634),NHE2(Q9UBY0) and NHE3(P48764) are shown. Sequences were aligned withClustalX tities are shaded and the arrowhead indicates the predictedsignal peptide cleavage site. The last translated amino acid of eachpbo-4 mutant allele is denoted by a star. The black bars indicatepredicted transmembrane domains, and the dotted line denotes there-entrant loop as determined for NHE1. The boxed sequence indicates thepredicted calmodulin binding domain. The positions of the GFP insertionsare indicated by triangles.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. For example, reference to “a host cell” includes a pluralityof such host cells.

As used herein a “Substantially Identical” polypeptide sequence means anamino acid sequence which differs from a reference sequence only byconservative amino acid substitutions, for example, substitution of oneamino acid for another of the same class (for example, valine forglycine, arginine for lysine, etc.) or by one or more non-conservativesubstitutions, deletions, or insertions located at positions of theamino acid sequence which do not destroy the function of the polypeptide(assayed, for example, as described herein). Preferably, such a sequenceis at least 80-100%, more preferably at least 85%, and most preferablyat least 90% substantially identical at the amino acid level to thesequence used for comparison.

To identify the nature of the signal, source, and target, screens formutations affecting the defecation cycle and in particular, mutationsspecifically affecting the posterior body contraction have beenperformed. From these screens, only three genes, pbo-1, pbo-4 and pbo-5result in a specific loss of posterior body contraction, withoutaffecting any other aspect of the defecation motor program. The pbo-1gene has not been cloned. The pbo-4 gene encodes a putative Na⁺/H⁺exchanger and is required for activation of the posterior bodycontraction (P. Dal Santo, personal communication; see Discussion). Itsexpression in the posterior intestine suggests that pbo-4 functions as amediator between the calcium signal of the clock in the intestine andthe muscle receptor that stimulates the posterior body contraction.

Na⁺/H⁺ exchangers have been implicated in numerous physiologicalprocesses such as intracellular pH homeostastis, cell volume regulation,and reabsorption of NaCl across epithelial cells. However, the protoncan be involved in more complex cellular processes other thanintracellular pH regulation. Deletion of genes involved in H^(|)secretion and reception results in a broad range of cellular andbehavioral phenotypes. Significantly, deletion of the murine NHE1 genereveals it is required for functions as diverse as pH homeostasis tocell morphology and adhesion. Consistent with a broader role for protonsas intercellular messengers, a large family of proton-gated channels,termed the acid-sensing ion channels (ASICs), have been identified.Knockouts of specific ASIC family members have demonstrated that properH^(|) signaling is required not only for sensory modalities such asnociception and mechanoreception, but also for synaptic plasticity andlearning and memory in the brain. While specific H⁺-gated receptors suchas ASIC1 are expressed in hippocampal neurons, the source of protonsrequired to activate these receptors remains a mystery. Hence, thepresent invention is useful in the field of medicine and providesvaluable research tools for the identification of medical and veterinarycompounds.

The gene pbo-5 gene encodes a novel H⁺-gated ion channel subunit. Whenexpressed in Xenopus oocytes, PBO-5 co-assembles with the PBO-8 subunitto form a functional H⁺-gated nonselective cation channel. PBO-5 andPBO-8 are expressed in the posterior body wall muscles, suggesting thesetwo subunits are localized to the appropriate tissues to mediateposterior body contraction. The channel encoded by PBO-5/8 is aninwardly-rectifying non selective cation channel. The molecularidentification of the PBO-5 and PBO-8 receptor subunits defines a uniqueclass of the cys-loop ligand-gated ion channel superfamily, anddemonstrates a non-neuronal signaling mechanism. Furthermore, thefunctional characterization of H⁺-sensitivity demonstrates that thePBO-5/8 signaling pathway defines a novel mechanism of cellularcommunication that relies on H⁺ as a physiological transmitter.

Methods

Molecular Characterization of pbo-5 and pbo-8

The sequence of the pbo-5 transcript was determined by reversetranscription of wild-type RNA followed by PCR amplification (RT-PCR)and direct sequencing. The 5′ trans-spliced sequence was obtained by PCRamplification using an SL1 primer and a nested, gene specific primer inthe second exon. The 3′ sequence was obtained by PCR amplification withnested primers in the third predicted exons and with an oligo-dT primer.PCR products were cloned into the pCR2.1 TA cloning vector (Invitrogen)and a full length clone pPD68 was generated.

PBO-8 cDNAs were isolated by RT-PCR. We determined the 5′ end of thegene by circular RACE (Maruyama et al., 1995). The 3′ end of the genewas based on computer prediction and sequence similarity to PBO-5. Toisolate full-length cDNAs, oligonucleotide primers were designed to the5′ and 3′ untranslated regions of pbo-8. PCR products were cloned intopCR2.1, and subsequent products were sequenced to generate a full-lengtherror free PBO-8 cDNA.

Electrophysiology of PBO-5 and PBO-8

To generate plasmid constructs for Xenopus oocyte expression, the foillength error-free cDNA was subcloned into the pSGEM expression vector(courtesy M. Hollmann). The pbo-5 expression vector was constructed bycutting the pPD70 plasmid with restriction enzymes SacII and Eag1, thenusing T4 DNA polymerase to blunt the ends. Cut products were gelpurified (Qiagen) and re-ligated using T4 DNA ligase (Promega) to createthe plasmid pAB20. The PBO-8 expression construct was made by cutting apreviously sequenced error-free cDNA (M.

Peter, unpublished). The cDNA was first cloned into the pSGEM expressionvector. Next, the restriction enzymes SacII and BsaBI were used to cutthe plasmid, and the ends were blunted with T4 DNA polymerase. Cutproducts were gel purified and re-ligated to produce pAB21.

Capped RNA was prepared using the T7 mMessage mMachine kit (Ambion).Xenopus oocytes were collected and coinjected with 25 ng each of PBO-5and PBO-8 cRNA and two-electrode voltage clamp recordings were performed3-5 days post-injection. The standard bath solution for dose-responseand control I-V experiments was Ringer's (in mM): 115 NaCl, 1.8 BaCl2,10 Bis-Tris Propane (pH 7.4 Acetic acid). For dose-response experiments,each oocyte was subjected to a 5 second application of test pH (7.0-5.0)with 2 minutes of pH 7.4 wash between test applications.

Ion selectivity experiments: All points are responses to test pulseapplications of pH 6.8 for 5 seconds. The reversal potential ofPBO-5/PBO-8 expressing oocytes were first determined in standardRinger's (in mM): 115 NaCl, 1.0 CaCl2, 10 Bis-Tris Propane (pH 7.4,acetic acid). For chloride permeability a simplified solution was used,chloride-free (in mM): 115 Na⁺ gluconate, 1.0 CaCl₂, 10 Bis-Tris Propane(pH 7.4 acetic acid). For Na⁺ permeability a simplified solution wasused, Na⁺ free (in mM): 115 mM N-methyl D-glucamine (NMDG), 1.8 CaCl₂,10 Bis-Tris Propane (pH 7.4 acetic acid)). All solutions were brought topH by addition of NaOH or acetic acid. To determine K⁺ permeability, I-Vexperiments were performed in K⁺ Ringer's (in mM): 115 K⁺-gluconate, 1.0CaCl₂, 10 Bis-Tris Propane (pH=7.4 acetic acid). To determine Ca²⁺permeability extracellular Na⁺ was lowered to 90 mM and extracellularCaCl₂ was increased to 3 mM and 10 mM, compare to 1 mM Ca²⁺ control. Tobetter resolve calcium permeability, the Na⁺ free solution was usedexcept CaCl₂ was increased ten-fold, 18 mM CaCl2 (in mM): 115 NMDG, 18CaCl₂, 10 Bis-Tris Propane, (pH 7.4 acetic acid). Osmolarity wasmeasured for each solution and was maintained at 240 mOsm by theaddition of sucrose. All recordings were done at room temperature. Weused 3M KCl filled electrodes with a resistance between 1-3 MΩ. We useda 3M KCl agar bridge to minimize liquid junction potentials, and allliquid junctions potentials arising at the tip of the recordingelectrode were corrected online.

Data analysis. Data acquisition and analysis were performed usingAxograph (Axon Instruments) software, and curve fitting and statisticalanalysis were performed with Prism (Graphpad). Dose-response curves fromindividual oocytes were normalized to the maximum and minimum values andaveraged for at least eleven oocytes. Normalized data were fit to thefour-parameter equation derived from the Hill equation:Y=Min+(Max−Min)/(1+10(LogEC50−X)(nH)), where Max is the maximalresponse, Min is the response at the lowest drug concentration, X is thelogarithm of agonist concentration, EC50 is the half-maximal response,and nH is the Hill coefficient. Error bars represent the standard errorof the mean.

Reversal potential values represent averaged linear regressionmeasurements of individual experiments above and below the point ofX-axis intersection. Because the PBO-5/PBO-8 receptor exhibitsrectification we used linear regression from −15 to +15, where therelationship is most linear, to determine the reversal potential. Errorbars represent the standard error of the mean.

Results

pbo-5 Behavioral Analysis

Recessive alleles in the pbo-5 gene result in a strong posterior bodycontraction defective (Pbo) phenotype. The posterior body contraction iscompletely absent from these mutants while the other aspects ofdefecation such as anterior body contraction, enteric musclecontraction, and cycle timing remain unaffected. Other behaviorsincluding locomotion, egg laying, feeding and mating are also normal.

In contrast to the recessive pbo-5 alleles, two dominant mutations,n2331 and ox7, result in a distinctive hypercontracted phenotype knownas posterior body cramp. Specifically, when the defecation cycle isinitiated in these mutants, the posterior body muscles contract but failto immediately relax, and the enteric muscle contraction occurs whilethe posterior body wall muscles are still contracted. As the animalsmove, the posterior body muscles eventually relax and the next cycle canbe observed.

pbo-5 Cloning

The pbo-5 gene was mapped to the right arm of chromosome V and cloned(FIG. 2). The Y44AE predicted open reading frame encodes pbo-5,confirmed by mutant allele sequencing (Table 1). Furthermore, amini-gene construct containing the pbo-5 cDNA fused to 4.4 kb ofsequences upstream of the second exon rescues the phenotype ofpbo-5(ox24) mutants.

TABLE 1 pbo-5 mutations

pbo-5 Encodes a Ligand-Gated Ion Channel

The primary structure of the pbo-5 cDNA was determined by reversetranscription and polymerase chain reaction. The pbo-5 cDNA includes anSL1 trans-spliced leader sequence at the 5′ end and a total of 9 exonsspanning a 7 kb genomic region (FIG. 2). The pbo-5 mutations fall intofour categories: (1) nine deletions (2) three nonsense (3) sevenmissense, and (4) one splice junction mutation (Table 1). The predictedpbo-5 cDNA encodes a 499 amino acid protein (FIG. 3).

BLAST and protein motif queries of PBO-5 suggest that it is a member ofthe ligand-gated ion channel superfamily. Ligand-gated ion channels areoligomeric receptor complexes containing an integral ion channel that isopened upon ligand binding (Betz, 1990). Ion channels that are gated byacetylcholine, serotonin, y-aminobutyric acid, and glycine are asuperfamily of homologous neurotransmitter receptors, termed thecys-loop superfamily. Although over 100 ligand-gated ion channelsubunits have been identified in numerous organisms, each subunit can beassigned to one of the four receptor families based on sequencesimilarity, and/or functional activity. All ligand-gated ion channelsubunits share a common structural and functional homology and have ahigh degree of amino acid similarity. The subunits are demarcated by asignal peptide, an extracellular amino-terminus that contains consensusligand binding sites and an invariant disulfide bonded loop (cys-loop),four transmembrane domains (M1-M4), a large cytoplasmic loop betweenM3-M4, and a short extracellular carboxy terminus (Karlin and Akabas,1995; Ortells and Lunt, 1995) (FIG. 2). Electron microscopy,electrophysiological and structural data suggest that ligand-gated ionchannels are formed from five homologous subunits that arepseudo-symmetrically arranged around a central ion channel, such thatthe M2 domain lines the ion-channel wall and determines ion selectivity(Betz, 1990; Brejc et al., 2001; Unwin, 1993).

Hydropathy plots and alignment of PBO-5 with various ligand-gated ionchannel subunits demonstrates that PBO-5 contains the hallmarks ofligand-gated ion channels (FIG. 3). Additionally, PBO-5 contains manyresidues that underpin the conserved secondary structure of the cys-loopligand-gated ion channel superfamily (Brejc et al., 2001). BLASTsearches of PBO-5 against human, mouse, Drosophila, and C. elegansgenomes suggest that PBO-5 most closely resembles cholinergic receptors.However, phylogenetic analysis demonstrates that

PBO-5, and related orthologs, represents a divergent subunit that cannotbe categorized into one of the four families based on sequencesimilarity (FIG. 4). BLAST searches against the C. elegans genomerevealed another predicted protein, PBO-8, which contained high sequenceidentity to PBO-5. For example, accession numbers P12389, 008952 andQ7T2T8. Based on sequence similarity and residues known to be involvedwith specific ligand sensitivity, the ligand for the PBO-5 receptor wasnot clearly identifiable from the native sequence. Further, mutants thatare defective in GABA, acetylcholine, serotonin, and peptidergicneurotransmission do not exhibit posterior body contraction defects.Therefore, it seemed unlikely that PBO-5 would be activated by knownclassical neurotransmitters.

pbo-5 is Expressed in the Posterior Body Wall Muscles

If PBO-5 is the receptor that mediates posterior body contraction thenit should be expressed in the body wall muscles. To determine thecellular expression of pbo-5, a transcriptional pbo-5::gfp fusion genewas constructed that contained 3.8 kb upstream sequence of thetranslational start codon fused to the GFP open reading frame (Chalfieet al., 1994). Stable chromosomally integrated lines of this constructexpressed GFP in the most posterior muscle cells of the tail and in asmall number of neurons in the head (FIG. 5). The expression pattern ofPBO-5 demonstrates that it is expressed in the appropriate cells tomediate posterior body contraction.

PBO-5 is not Activated by Classical Neurotransmitters

To determine if PBO-5 can form a homo-oligomeric receptor, we injectedPBO-5 cRNA in Xenopus oocytes. Since sequence information did notconfidently predict the ligand that would activate the putative PBO-5receptor, a candidate approach was undertaken to ascertain the ligand.Two-electrode voltage clamp recordings were used to assay receptorfunctionality. A host of classical neurotransmitters such asacetylcholine, choline, GABA, glycine and serotonin were applied toPBO-5 injected oocytes, yet all ligands failed to elicit a functionalresponse.

Typically, muscle type acetylcholine receptors require both α and non-αsubunits to form functional receptors. The predicted PBO-8 protein isthe most closely related to PBO-5 of all predicted cholinergic-likereceptors in C. elegans (FIG. 4).

PBO-8 Primary Structure and Expression

The pbo-8 predicted open reading frame encodes a protein that ishomologous to ligand-gated ion channels (FIG. 3). Significantly, thePBO-8 protein most closely resembles PBO-5 (FIG. 4). To determine theprimary structure of PBO-8 RT-PCR was performed and full-length cDNAclones isolated. The PBO-8 full-length cDNA consists of 13 exons thatspan 4.1 kb of genomic DNA (FIG. 6A). The PBO-8 cDNA encodes a 423 aminoacid protein (FIG. 6A). We performed protein alignments with PBO-8 andPBO-5 revealing they share 35% identity. With the exception of oneconservative amino acid change, the residues in the M2 domain areidentical (FIG. 3). If PBO-8 oligomerizes with PBO-5 to form afunctional receptor, then PBO-8 should have overlapping expression withPBO-5. To address the expression pattern of PBO-8, a transcriptionalfusion containing 4 kb of upstream promoter sequence fused to GFP wasconstructed. GFP expression was observed in the most posterior body wallmuscles in the tail, identical to PBO-5 expression (FIG. 6B). These datasuggest that PBO-8 may oligomerize with PBO-5 to form a functionalreceptor.

PBO-5 /PBO-8 Forms a if-Gated Ion Channel

To determine if PBO-5 and PBO-8 can co-assemble to form a functionalreceptor, we injected PBO-5 and PBO-8 cRNA into Xenopus oocytes.Agonists which function at other ligand-gated ion channels (such as,ACh, GABA, glycine, 5-HT, glutamate, and choline) lacked the ability toactivate PBO-5 homomers, PBO-8 homomers or PBO-5/PBO-8 heteromers.

Activation of receptors was not unexpected because genetic evidencedemonstrates acetylcholine, GABA, glutamate and serotonin are notrequired for posterior body contraction. To determine the possiblesignal that mediates posterior body contraction we examined the pbo-4gene. The predicted pbo-4 gene encodes a protein related to Na⁺/H⁺exchangers. PBO-4 is expressed in the posterior intestine and isrequired for posterior body contraction. Na⁺/H⁺ exchangers mediate theexchange of one Na⁺ into the cell for one H⁺ out of the cell. Theexpression of PBO-4 in the posterior intestine parallels the expressionpattern of PBO-5 and PBO-8 in the posterior body wall muscles. Thesedata suggest that PBO-4 mediates the secretion of H⁺ from the intestine,into the pseudocoelomic space, adjacent to PBO-5/PBO-8 posterior muscleexpression. Therefore, without wishing to be bound by theory, wehypothesized that H⁺ ions may activate or may be required forco-activation of PBO-5/PBO-8 receptors.

To determine if H^(|) ions are sufficient to activate PBO-5/PBO-8receptors, we applied test pulses of varying pH to PBO-5/PBO-8expressing cells. Test pulses of pH 6.8 evoked robust inward currents,indicating that ⁺ ions are sufficient to activate recombinant receptors(FIG. 7, inset). To demonstrate that current responses evoked by changesin pH were not due to endogenous channels or transporters, we appliedmaximal test pulses of pH 5.0 to water injected and uninjected oocytes.Only oocytes injected with PBO-5/PBO-8 cRNA exhibited H⁺-gatedresponses. To determine if PBO-5 and PBO-8 can form functional homomericH⁺-gated ion channels, we injected each subunit alone into oocytes.Injection of PBO-5 or PBO-8 cRNA into oocytes, resulted in little or nofunctional expression compared to oocytes co-injected with PBO-5 andPBO-8 cRNA. These data suggest that the PBO-5/PBO-8heteromultirnerization is required for efficient functional receptorexpression in vitro.

H⁺ ions have been demonstrated to modulate classical synaptictransmission. For example, acidic changes in pH inhibit acetylcholinereceptor function, where alkaline environments enhance receptor function(Palma et al., 1991; Pasternack et al., 1992). To determine whether ornot classical neurotransmitters co-activate PBO-5/PBO-8 receptors, weapplied pH 6.8 with and without 1 mM ligand. Application of pH 6.8 plusacetylcholine, choline or GABA was not significantly different from pH6.8 only application (data not shown). Taken together, these datasuggest that H⁺ ions alone are sufficient to fully activate PBO-5/PBO-8receptors.

To determine the pHSO (half-maximal activation) of PBO-5/PBO-8heteromultimers, we first identified the pH range at which therecombinant receptors were activated. We determined that the PHIO (10%maximal activation) was approximately pH 7.0. We set our perfusionbuffer at pH 7.4 for all experiments, where no activation of recombinantreceptors was observed. A pH₅₀=6.83±0.01 was determined by applyingdecreasing pH test pulses (pH 7.0-5.0). A steep Hill coefficient of9±0.66 was determined, demonstrating PBO-5/PBO-8 receptors exhibitsignificant H⁺ binding cooperativity (FIG. 7).

PBO-5/PBO-8 Ion-Selectivity

Current-voltage (I-V) analysis was used to characterize the ionicconductance underlying PBO-5/PBO-8 responses. The H-gated current had areversal potential of 10.18±0.80 mV in Ringer's solution (FIG. 8A). TheI-V relationship demonstrates that PBO-5/PBO-8 exhibits inwardrectification (FIG. 8A, inset). The positive reversal potential suggeststhat PBO-5/PBO-8 encodes a cation-selective ion channel. To determine ifNa⁺ underlies PBO-5/PBO-8 H⁺-evoked responses, we replaced Na⁺ with thelarge cation N-methyl D-glucamine. In Na⁺-free solution, the inwardcurrent was eliminated and the reversal potential was −82.90±8.13 mV(FIG. 8B). Furthermore, to demonstrate anions such as Cl⁻ do not flowthrough PBO-5/PBO-8 channels, we replaced extracellular Cl⁻ with theimpermeable anion gluconate. In C⁻ free solution, the inward currentremained with no significant shift in reversal potential (ΔE_(rev)=0.25mV, P>0.05, two-way ANOVA), compared to control (FIG. 8C). Thisdemonstrates Na⁻ is the primary charge carrier through PBO-5/PBO-8channels.

To determine the cation-selectivity of the PBO-5/PBO-8 ion channel, wesubstituted extracellular Na⁺ with equivalent K. In K⁻ Ringer'ssolution, I-V relationships were not significantly different (P>0.05two-way ANOVA) compared to Na⁺ Ringers control (FIG. 8D). Additionally,strong inward rectification was present in I-Vs determined in K^(|)Ringers, demonstrating rectification was not due to ion-selectivity.Taken together, these data demonstrate PBO-5/PBO-8 encodes anon-selective inwardly rectifying cation channel.

Next, Ca²⁺ permeability of the PBO-5/PBO-8 ion channels was assayed. TheI-V relationships under three Ca²⁺ concentrations: 1 mm control, 3 mM,and 10 mM, were determined. Increasing calcium to 10 mM resulted in apositive shift in reversal potential from control (FIG. 9A). To betterresolve calcium permeability, NaCl was replaced with NMDG in theextracellular solution, making Ca²⁺ the only relevant extracellular ion.Increasing Ca²⁺ in this solution revealed that PBO-5/PBO-8 channels arecalcium permeable. Increasing Ca²⁺ 10 fold caused a +50 mV shift inreversal potential compared to control (FIG. 9B).

Interestingly, H⁺-evoked inward currents were significantly reduced asextracellular Ca²⁺ was increased (FIG. 9). Specifically, a control pulseof pH 6.8 with 1.0 mM Ca²⁺ evoked a large response, and an equivalenttest pulse of pH 6.8 with 10.0 mM Ca²⁺ evoked a much smaller response.This suggests that, while Ca²⁺ is permeable, high amounts of Ca²⁺inhibit gating of PBO-5/PBO-8 channels.

To further investigate the Ca^(2|) inhibition of PBO-5/PBO-8 receptors,dose response experiments were performed under different extracellularCa²⁺ conditions (0.1, 1, 3, 10 mM). PBO-5/PBO-8 receptors under 1 mMCa²⁺ control conditions exhibited a pH₅₀=6.88 and a Hill coefficient of6 (FIG. 10A). As Ca²⁺ was increased to 10 mM a right shift in theactivation curve of PBO-5/PBO-8 was observed. Specifically, asignificantly different pH₅₀=6.59 and a Hill coefficient of 4 wasdetermined under 10 mM Ca²⁺ test conditions (FIG. 10A). These datademonstrate that as extracellular Ca^(2|) is increased, H^(|)sensitivity and cooperativity decreases.

Next, to determine if increasing extracellular Ca²⁺ caused an inhibitionof maximal PBO-5/PBO-8 activation, Ca²⁺ conditions at three different pHranges (7.0, 6.8, and 6.0) were applied. H-evoked responses weresignificantly reduced, compared to 1 mM Ca²⁺ control, at pH 7.0 and 6.8as Ca²⁺ was increased to 3 mM and 10 mM (FIG. 10B). However, at pH 6.0,increasing extracellular Ca²⁺ had no effect on maximal activation ofPBO-5/PBO-8 (FIG. 10B). These data suggest that Ca²⁺ acts as acompetitive antagonist at PBO-5/PBO-8 receptors. We are furtherexamining the mechanism of Ca²⁺ antagonism to PBO-5/PBO-8 receptors.

Discussion

It is demonstrated that pbo-5 encodes a novel ligand-gated ion channelsubunit required to initiate the posterior body contraction of thedefecation cycle. Loss of pbo-5 activity specifically eliminates theposterior body contraction, while gain-of function alleles result inhypercontraction of the posterior body muscles. PBO-5 is expressed inthe most posterior muscle cells of the tail, suggesting it is properlylocalized to mediate the posterior body contraction. Finally, it isdemonstrated that PBO-5 heteromultimerizes with PBO-8 to form a novelH⁺-gated ion channel when expressed in Xenopus oocytes. The channelencoded by PBO-5/PBO-8 is a nonselective inward rectifying cationchannel. The molecular identification and functional characterization ofthe PBO-5 and PBO-8 receptor subunits defines a novel group of thecys-loop ligand-gated ion channel superfamily. Furthermore, thefunctional characterization of H^(|)-sensitivity demonstrates that thePBO-5/PBO-8 signaling pathway defines a novel mechanism of cellularcommunication.

H⁺ Dependence of PBO-5/PBO-8

The overlapping cellular expression of PBO-5 and PBO-8 in the mostposterior body muscles of that tail, coupled with the fact thatfunctional receptor expression is only observed when both PBO-5 andPBO-8 are coinjected into oocytes, strongly suggests the PBO-5/PBO-8represents the native receptor that mediates posterior body contraction.We have demonstrated that H⁺ ions are sufficient to activate PBO-5/PBO-8receptors.

The defecation cycle in a wild-type animal occurs every 50 seconds withlittle variability and the first muscle contraction to occur is theposterior body contraction. It has been demonstrated that periodiccalcium release in the intestine correlates with the onset of theposterior body contraction (Dal Santo et al., 1999). The itr-1 genewhich encodes an inositol triphosphate (IP₃) receptor is required in theintestine for proper defecation cycle timing (Dal Santo et al., 1999).Hypomorphic itr-1 alleles exhibit long or no defecation cycle cycles(>50 seconds), while overexpression of itr-1 results in short defecationcycle times (<50 seconds). Furthermore, calcium imaging of itr-1hypomorphic alleles that have no defecation cycles (i.e., no posteriorbody contraction) fail to exhibit calcium oscillations (Dal Santo etal., 1999). Taken together, these data suggest that IP₃ receptoractivity in the intestine is required for the signaling of each musclecontraction in the defecation cycle.

The posterior body contraction is the initial contraction in thedefecation motor program, and does not rely on neuronal input. ThePBO-5/PBO-8 receptor is gated by H⁺ ions, and PBO-5 is required forposterior body contraction. It has been demonstrated that IP₃ receptor(itr-1) mediated calcium signaling in the intestine is required for theactivation of the posterior body contraction (Dal Santo et al., 1999).However, the nature of the signal was unknown as mutants with defects inclassical neurotransmission have normal posterior body contraction.

The pbo-4 gene encodes a protein that has similarity to Na⁺/H⁺exchangers (NHEs). pbo-4 mutants are phenotypically identical to pbo-5mutants, and the pbo-4; pbo-5 double mutant is identical to both singlemutants. This suggests that pbo-4 and pbo-5 are in the same signalingpathway that mediates posterior body contraction.

Furthermore, the expression pattern of pbo-4 is restricted to theposterior intestine, adjacent to the muscle expression of PBO-5 andPBO-8. Na⁻/H⁺ exchangers have been implicated in a number of processesincluding intracellular pH and cell volume regulation, and inreabsorption of NaCl across epithelial cells (Counillon and Pouyssegur,2000; Orlowski and Grinstein, 1997). Plasma membrane NHEs mediate theelectroneutral exchange of Na⁺ ions into the cell for H⁺ ions out,thereby acidifying the extracellular environment while increasingintracellular pH. Mammalian NHEs are regulated by a number of factorsincluding phosphorylation, calcium, and by interactions with accessoryproteins (Orlowski and Grinstein, 1997). For example, mammalian NHE1 issensitive to increases in cytosolic Ca⁻, due to a high-affinitycalmodulin binding site present on the C-terminal tail of the protein(Bertrand et al., 1994). Deletion of this binding site causes NHE1 to beconstitutively active (Wakabayashi et al., 1994; Wakabayashi et al.,1997). Therefore, under normal conditions (low Ca²⁺), thecalmodulin-binding site is unoccupied and exerts an inhibitory effect.An intracellular rise in Ca²⁺ stimulates Ca²⁺/calmodulin binding toNHE1, thereby releasing inhibition, and activating Na⁺/H⁺ exchange.Analysis of the C-terminal tail of the PBO-4 protein predicts a putativecalmodulin binding site as well as a consensus phosphorylation site forCaMKII.

The structural analysis and expression of pbo-4 in the intestinepredicts that PBO-4 mediates the exchange of Na⁺ ions into the intestinefor H⁺ ions out of the intestine into the pseudocoelomic space. H⁺binding has been demonstrated herein to be sufficient to activatePBO-5/PBO-8 receptors. Therefore, without wishing to be bound by theory,it is believed that the H⁺ signal arises from the intestine and H⁻transport is mediated by PBO-4.

It has been demonstrated that IP₃ receptor mediated release of calciumfrom the intestine correlates with the onset of the posterior bodycontraction and is required. Therefore, it is believed that a necessaryfeature of the posterior body contraction signaling mechanism is that itbe modulated by calcium. The pbo-4 gene, expressed in the posteriorintestine, encodes a Na⁺/H⁺ exchanger with a predicted Ca²⁺/calmodulinbinding domain. It has now been demonstrated that H⁺ ions are sufficientto activate PBO-5/PBO-8 receptors (e.g., using Xenopus oocytes (invitro)) that are localized to the posterior body muscles.

Without wishing to be bound by theory, from this data it is proposedthat for posterior body contraction: 1) IP₃ receptors localized to thesmooth endoplasmic reticulum in the intestine are activated every 50seconds. Activation results in the intracellular rise of Ca²⁺ in theintestine; 2) Ca²⁺ binds to the C-terminal calmodulin-binding domain ofPBO-4, thereby activating H⁺ transport activity; 3) PBO-4 transports H⁺ions out of the intestine acidifying the pseudocoelomic space; and 4) H⁺ions bind and activate the PBO-5/PBO-8 receptors expressed in posteriorbody wall muscle, thereby allowing Na⁺ influx into the cell causing adepolarization and subsequent muscle contraction (FIG. 11).

This simple model can account for the non-neuronal nature of theposterior body contraction, and involves strict calcium regulation. Thepresent invention demonstrates that PBO-5/PBO-8 receptors areexquisitely sensitive to changes in extracellular pH, as determined inheterologous cells (pH₅₀=6.83±0.01). The sensitivity of PBO-5/PBO-8receptors suggests minor acidification of the pseudocoelomic space isrequired to activate the posterior body contraction. The pseudocoelomicspace separates the intestine from the muscle, and is relatively smallas observed in electron micrographs. Thus, the acidification of thepseudocoelomic space at the posterior end of the animal to a pH ˜6.8 isnot physiologically unreasonable. In vivo recording from the posteriorbody wall muscles is preformed to recapitulate the in vitro findings.

PBO-5/PBO-8 is a Novel Cys-Loop Ligand-Gated Ion Channel

The PBO-5 and PBO-8 subunits display the structural motifs common to thesuperfamily of cys-loop ligand-gated ion channels. However, thedistinctive sequence and functional activity prevent classificationwithin any of the four established receptor families. Motor behaviors,such as muscle contraction, are typically controlled via fast synaptictransmission between a motor neuron and muscle cell. The presentinvention demonstrates that H⁺ ions alone are sufficient to activaterecombinant receptors and that co-application of other classicalneurotransmitters has little effect on activation. Furthermore, thefunctional characterization of the receptor and proposed model ofexcitation provide a unique form of fast non-synaptic communication.Protons have been demonstrated to be modulatory at “cys-loop”ligand-gated ion channels; however, this is the first evidence thatprotons can directly activate this receptor subclass. Therefore, thepresent invention demonstrates that H⁺ fulfills the criteria of a fasttransmitter in C. elegans.

In one exemplary embodiment mammalian paralogs and/or orthologs areidentified, for example by BLAST searches using PBO-5 and PBO-8sequences (e.g. SEQ ID NOS: 6 and 7), and one or more of the paralogsand/or orthologs is confirmed to be a component of a H⁺-gated ionchannel. In particular, there are a number of predicted cholinergic-likeproteins to which PBO-5 and PBO-8 share identity. Changes inextracellular pH have been demonstrated to regulate ion channelfunction, hi most cases, H⁺ ions modulate classical neurotransmission byinhibiting or exciting various classical ligand-gated ion channels.While it has been demonstrated that H⁺ signaling is a major pathway forpain sensation through the acid-sensing ion channels (ASIC), direct H⁺signaling has never been associated with cys-loop ligand-gated ionchannels (Waldmann et al., 1997; Waldmann et al., 1999).

Recently, a novel Zn^(2|) activated ligand-gated ion channel (ZAC) hasbeen cloned and characterized in humans (Davies et al., 2003). The ZACchannel represents a distinct class of cys-loop ligand-gated ionchannels that is not activated by classical neurotransmitters.Interestingly, the ZAC subunit has been retained in some mammals (humanand dog), but has been lost by others (mouse and rat), demonstratingspecific cell signaling mechanisms may vary across species. Therefore,an exemplary embodiment of the invention provides a method for theidentification and characterization of the H^(|) gated PBO-5/PBO-8receptor in other species.

In an exemplary embodiment, the pharmacology of the PBO-5/PBO-8 receptoris determined, in another exemplary embodiment modulation of PBO-4 byCa²⁺ is determined. In yet another exemplary embodiment, isolation ofPBO-8 mutants is performed and the mutant phenotype assayed relative toPBO-5 and/or PBO-4. Optionally, genetic analysis is conducted todetermine if PBO-8 is in the same genetic pathway as PBO-5 and/or PBO-4.

To determine if H⁺ ions are required to initiate a posterior bodycontraction endogenous acidification of the pseudocoelomic space isblocked. For example, 100 mM Bis-Tris Propane buffered saline (pH 7.2)is injected into the pseudocoelomic space of an animal and defecationcycles postinjection observed. It is believed that animals that have hadbuffered saline injected into the pseudocoelomic space will fail toinitiate a posterior body contraction while all other steps of thedefecation cycle will be normal. The injected animals should phenocopyboth pbo-4 and pbo-5 mutants.

Alternatively, a caged buffer is used to assay the requirement for H⁺activation of PBO-5/PBO-8 receptors in vivo. For example, animals areinjected with a caged buffer (Molecular Probes) and the buffer uncaged,by flash photolysis, as a posterior body contraction is about to occur.An advantage of the latter scheme is that an animal can be injectedfirst and then observed for a number of cycles to determine if itretains normal defecation cycles, and in particular posterior bodycontractions. After quantification of a number of defecation cycles, thebuffer is uncaged and the animal observed for a specific loss ofposterior body contraction. Li addition, the inverse experiment, wherecaged protons are injected, may be conducted to elicit an out of phaseposterior body contraction by uncaging the protons during theintercycle.

Functional Analysis of Dominant pbo-5 Alleles

Several of the pbo-5 mutations provide particularly interesting insightsinto the structure and function of the PBO-5 protein. Loss-of-functionmutations cause an absence of posterior body contraction, whilegain-of-function mutations cause hypercontraction. Two dominant alleles(n2331 and ox7) result in the same mutation within the M2 domain of theion channel. Specifically, residue 316 is mutated from leucine tophenylalanine (L316F). The M2 domain lines the ion channel wall, andresidues in and around M2 affect ion selectivity and receptordesensitization (Leonard et al., 1988). An adjacent residue is alteredin the M2 domain of the cholinergic DEG-3 gain-of-function mutant, andcauses cell death (Treinin and Chalfie, 1995). It was proposed thatprolonged opening and increased ion flow through DEG-3 ion channelsunderlies the degeneration. Similarly, dominant gain-of-functionmutations within the M2 domain of acetylcholine receptors leads toincreased ion flow through slower channel closure (Engel et al., 1996).Therefore, it is believed that the cramp phenotype observed in thedominant alleles is due to a channel that has slower inactivation. Todetermine if pbo-5 (n2331dm) encodes a slowly inactivating receptor,cDNAs that carry the mutation are isolated and pbo-5 (n2331dm) cRNA iscoexpressed with wild-20 type PBO-8, and receptor function is assayed.It is believed that pbo-5 (n2331dm) will result in a response to acidicpH application that is prolonged and enduring, rather than desensitizingas in the wild-type.

PBO-5/PBO-8 Pharmacology

PBO-5/PBO-8 forms a heteromultimeric H⁺-gated cation channel andextracellular Ca²⁺ concentration affects PBO-5/PBO-8 pH sensitivity. ThePBO-5/PBO-8 receptor subunits are structurally related to theligand-gated ion channel superfamily, and sequence analysis andfunctional activity place the PBO-5/PBO-8 receptor into a previouslyunknown family (FIG. 4). hi an exemplary embodiment, H⁺-gated cationchannel receptors are identified and assayed for sensitivity to knownligand-gated ion channel agonists and antagonist. In addition, H⁺-gatedcation channel receptors are identified in a subject, for example, amammalian or vertebrate subject, such as a human, and assayed forfunctions described herein. Such mammalian, or vertebrate, receptors areassayed for drug interactions with have agonist, antagonist or blockingactivity.

For example, it was observed during electrophysiological investigationof PBO-5/PBO-8 that the buffer HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) in ourextracellular solution produced a very rapid rundown of the H⁺-evokedcurrent that could not be explained by poor oocyte health. An initialpulse of pH 6.8 HEPES buffered solution gave a robust inward current.However, subsequent application of pH 6.8 HEPES caused a markedreduction in inward current. Eventually, PBO-/PBO-8 receptors could berun-down to zero current with repeated pH 6.8 applications that was notreversible with time or supramaximal pH 5.0 application. Initially anextracellular ion replacement was conducted, since a divalent ion suchas Ca²⁺ could impart the current block. However, Ca²⁺ reduced solutionsstill exhibited use-dependent block, therefore, the buffer containingHEPES was substituted with Bis-Tris Propane(1,3-Bis[tris(hydroxymethyl)methylamino) propane or MES(2-(N-Morpholino)-ethanesulfonic acid.

When HEPES was replaced with either Bis-Tris Propane or MES, repeatedacidic pH test pulses did not cause current run-down. Therefore, HEPESwas shown to be inhibitory to the receptor. Furthermore, HEPES wasdemonstrated to be the relevant blocking molecule by applying pH 6.8pulses of HEPES buffered solution until the evoked current was run downto zero. Following pulsed run down, a pH 6.8 pulse of Bis-Tris propanewas applied to the run down receptors. Application of pH 6.8 Bis-TrisPropane or MES buffered solutions to rundown receptors, evoked a robustinward current. Specifically, a current equal to or greater than theinitial pH 6.8 HEPES response was obtained.

How can HEPES be inhibiting the channel? Recently, the structure of anacetylcholine binding protein (AChBP) that is homologous to theN-terminus of acetylcholine receptors has been resolved (Brejc et al,2001). The structure of this protein agrees with the predictedN-terminal structure of ligand-gated ion channels. Importantly, it wasdetermined that the ligand binding sites are located at each of the fivesubunit interfaces, which are located in the extracellular N-terminalpart of the protein. Interestingly, the resolved crystal structurecontained a HEPES molecule present in each ligand-binding site. In thestructure it was determined that HEPES made no specific hydrogen bondwith the protein, but its quaternary ammonium group stacked onto Trp143making cation-it interactions (Brejc et al., 2001). These data suggestthat the HEPES molecule may be occluding the H⁺-binding site ofPBO-5/PBO-8 receptors. In an exemplary embodiment, mutations are made inand around the presumptive ligand-binding pocket and assayed forabolishment of HEPES inhibition.

As the PBO-5/8 receptor is exclusively activated by H^(|), it was nextdetermined if PBO-5/8 gating could be blocked with amiloride. Amilorideis a commonly used pharmacological agent used to block acid sensing ionchannels (ASIC). Application of 1 mm amiloride to PBO-5/8 expressingcells did not result current block, demonstrating the H-gating mechanismof PBO-5/8 receptors is different from the ASIC channels. Furthermore,common cholinergic antagonist such as d-tubocurare were applied toPBO-5/8 expressing cells, but all attempts to pharmacologically blockPBO-5/8 responses failed. These data suggest the gating mechanism ofPBO-5/8 receptors is quite different from both cholinergic and ASICreceptors.

In an exemplary embodiment, other compounds which bind the ligandbinding pocket, for example, of a vertebrate H⁺-gated channel, which maybe identified by the methods of the present invention, are assayed. Inone exemplary embodiment, agonists and/or antagonists are identified. Inanother exemplary embodiment, an agonist and/or antagonist ismanufactured as a medicament for the treatment of conditions relating toactivation or inactivation of the H⁻-gated channel.

PBO-4 Characterization

It has been demonstrated that initiation of the defecation cycle is ahighly calcium regulated process, and that calcium spikes in theintestine correlate with the onset of the posterior body contraction(Dal Santo et al., 1999). Therefore, it is believed that the defecationcycle regulated by calcium. The pbo-4 gene encodes a putative Na⁺/H⁺exchanger that is expressed in the posterior intestine. Without wishingto be bound by theory, it is believed that calcium release in theintestine, through IP₃ receptors, results in the activation of PBO-4.Specifically, PBO-4 contains a consensus calmodulin-binding domain thatis thought to be inhibitory to transport activity. Therefore, toactivate PBO-4, Ca²⁺ would bind the calmodulin site, thereby releasinginhibition and allowing proton transport, hi an exemplary embodiment,the secretion of H⁺ by PBO-4 from the intestine, in a calcium dependentmanner, PBO-4 is assayed for calcium binding and proton transportactivity.

The posterior body contraction occurs via a calcium-regulated mechanism.The cloned and characterized pbo-4 gene, which encodes a Na⁺/H⁺exchanger, is expressed on the basolateral surface of the posteriorintestinal cells, juxtaposed to the posterior body wall muscles.Utilizing caged protons, it has been demonstrated that acidification ofthe pseudocoelomic space, which separates the intestine from the bodywall muscles, was sufficient to activate the posterior body contractionin vivo. These results suggest that PBO-4 Na⁺/H⁻ transport activity isrequired to activate the posterior body contraction. Importantly, theidentification of the PBO-5/8 receptor confirms that H⁺ ions function asa primary transmitter in C. elegans. Furthermore, these resultsdemonstrate that Na⁺/H⁺ exchangers can function as a regulated H⁺release mechanism that mediates cellular signaling.

pbo-4 Mutations:

Allele Nucleotide Change Protein Change Sa300 GGA → AGA G318R n2658GGTATTAA(Δ548 bp) GACAC Deletion of the C-terminus ok583GCGACTCA (Δ1.702 Kb) aatttt Deletion of TM3- TM12 ok10 GCA → GAAA717D . . . 5 aa . . . Stop

PBO-8 Behavioral Characterization

In another exemplary embodiment, a deletion library is screened toidentify an allele, for example, an allele of pbo-8. Optionally, RNAitechnology may be used to knock out expression of PBO-8 or anortholog/paralog identified in another organism. Any assay known in theart may be used to study a PBO-4, PBO-5, and/or PBO-8 ortholog/paralog,for example, RNAi technology, see U.S. Patent Applications 20030153519,20030175772, 60/528,567, 60/518,856.

While this invention has been described in certain embodiments, thepresent invention can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of the invention using its generalprinciples. Further, this application is intended to cover suchdepartures from the present disclosure as come within known or customarypractice in the art to which this invention pertains and which fallwithin the limits of the appended claims.

All references, including sequence accession numbers, publications,patents, and patent applications, cited herein are hereby incorporatedby reference to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein.

REFERENCES

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1.-31. (canceled)
 32. A method for identifying a component of acation-selective ion channel from a vertebrate organism, comprising: (a)utilizing a library of vertebrate sequences; (b) contacting the libraryof vertebrate sequences with H^(|) at a pH range of 7.4 to 5.0; (c)assaying for cation exchange; (d) identifying a vertebratecation-selective ion channel responsive to increased acidity; and (d)confirming that ion channel is a cation-selective ion channel.
 33. Themethod of claim 32, further comprising isolating one or more componentsof the cation-selective ion channel.
 34. The method of claim 32, furthercomprising screening said component of said H⁻-gated channel for bindingto HEPES.
 35. A method of screening a candidate compound, comprising:(a) introducing a nucleic acid sequence encoding a sequence having atleast 80% identity to PBO-5 (SEQ ID NO: 2) or PBO-8 (SEQ ID NO: 3 or SEQID NO:4) into a host cell; (b) expressing the nucleic acid sequence toproduce a ligand-gated cation channel in the host cell; (c) contactingthe host cell with a candidate compound; and (d) screening foractivation or inhibition of the ligand-gated cation channel by thecandidate compound.
 36. The method of claim 35, further comprisingproducing the candidate compound.
 37. The method of claim 35, furthercomprising: (a) introducing a nucleic acid sequence encoding PBO-4 (SEQID NO:8) into a host cell; and (b) expressing the nucleic acid sequenceto produce a H⁺/Na⁺ exchanger in the host cell.